Peripheral Nervous System Regeneration
The fate of regenerating nerve fibers is determined by both intrinsic neuronal responses and extrinsic influences. Although the peripheral nervous system (PNS) is often regarded as regenerating well, long distance regeneration is the exception because Schwann cells and their scaffolding gradually disappear when their axons have died back. In addition, targets in muscle and skin may atrophy and no longer signal axon growth programs. The investigators seek a several-fold increase in the rate of extension without increasing axonal vulnerability or loss; strategies to enhance the growth-supporting properties of Schwann cells and maintain their viability; and tools to assess ongoing regeneration, especially imaging strategies that can be used in living mice and moved to humans.
One of the APNRR's collaborative efforts involves extrinsic influences on the PNS, including the roles of axonal regeneration inhibitors, growth factors, and Schwann cells. The PNS group brings together coordinated studies dealing with growth cone and axonal responses to the environment for regeneration (Larry Benowitz, John Griffin, Zhigang He, Ahmet Hoke, and Guo-li Ming), axon-Schwann cell interactions (Griffin, Hoke, Ming, Elior Peles), and new radiologic and physiologic assessments of in vivo nerve fiber growth (Kazim Sheik, Susumu Mori, Jeff Alger). Gene expression by transplanted stem cells as they differentiate toward Schwann cells and stem cell-based therapies are an added approach (Strickland, Heintz). Specifically, the PNS group focuses on promoting Schwann cell survival in the distal stump (Griffin, Hoke), the roles of axon-Schwann cell interactions in myelination in fostering nerve regeneration (Peles, Hoke, Griffin), the dual effects of MAG on axonal regeneration (Griffin, Ming, Jeff Twiss, Hoke, Roman Giger, Peles), the roles of NGF/TrkA signaling (Griffin, Hoke, Armin Blesch, Nathaniel Heintz), the role of growth factors in axonal retraction (Ming, Griffin), the heterogeneity of Schwann cells and the implications for specificity of regeneration (Hoke, Dan Geschwind), in vivo differentiation of stem cells toward Schwann cells (Sidney Strickland, Heintz), NAD-based strategies to promote axonal survival and speed regeneration (He, Hoke, Griffin, Ben Barres, Twiss), and myosin II antagonists to increase the rate of NGF mediated axonal regeneration (Fengquan Zhou, Griffin).
These efforts coincide with studies to understand and amplify intrinsic neuronal processes that result in a "growth state". Studies include posttranslational processes in injury and regeneration (Jeff Twiss) and gene expression studies (Mike Fainzilber) linked to models of spinal cord injury and dorsal and ventral root regeneration (Mark Tuszynski, Leif Havton). These experiments make use of the methodological advantages of the PNS, but much of the biology is shared with the central nervous system (CNS) models. APNRR investigators recognize that "PNS and CNS" are anatomically meaningful, but the distinctions are scientifically and organizationally constraining. Thus the PNS collaborations extend to models of the optic nerve and other CNS structures (Benowitz, He, and Ming).
Central Nervous System Injury Repair
The CNS Axonal Regeneration group identifies mechanisms that underlie the failure of axons to regenerate and methods to enhance regeneration by bringing together genetic, biochemical, cellular, and behavioral expertise. For instance, Michael Sofroniew, Barres and Benowitz examine how glial scarring influences the failure of axonal regeneration in a well-defined model system (optic nerve). Benowitz, Barres, Zhigang He, Fainzilber, and Twiss investigate intracellular signals that regulate axon growth including retrograde signals that inform the neuron of the state of its connections and the study of a protein kinase that may act as a "master switch" to control the expression of genes required for axon growth. Benowitz also investigates naturally occurring compounds that stimulate axonal plasticity after spinal cord injury and combinatorial treatments to further enhance growth. He, Barres, and Roman Giger examine possible molecular pathways involved in axon dieback degeneration after injury as well as the effect of anti-myelin monoclonal antibodies on injured CNS axons.
The nervous system does not respond well to injury and it is particularly vulnerable to disease. This is most apparent when axonal processes are disrupted and loss of communication between neurons results in permanent or near-permanent loss of function. To examine the nervous system's gene expression responses to injury that can set the stage for reparative processes, Twiss, Dan Geschwind, and Giovanni Coppola examine axonal mRNA profiles over time. The group of Tuszynski, Benowitz, Blesch, Fainzilber, and Geschwind are examining layer V motor cortex and primary dorsal root neurons for alterations in gene expression following mid-cervical spinal cord injury. A near term goal is to culture and regenerate pyramidal neurons (Tuszynski, Barres). To better understand the post-translational aspect of this regulation, Fainzilber and Twiss also investigate the products of axonal proteolysis and retrograde signaling in response to injury. In addition to axonal regulation in response to injury, this collaborative group also studies sensory neuron subtype expression (Yoram Groner, Nathaniel Heintz, Twiss), RNA localization/translation in retinal ganglion cell axon development (Barres, Fainzilber, Twiss), and RNA localization/translation in optic nerve regeneration (Benowitz, Fainzilber, Twiss). Future neural repair strategies will significantly benefit from a comprehensive understanding of these mechanisms. Here, we have brought together expertise in RNA transport and localization, translational control, signal transduction, proteomics (Al Burlingame and Ralph Bradshaw), and neural function after injury in a directed effort to apply post-transcriptional and post-translational regulatory mechanisms to neural repair strategies.
Injury to the lowest portion of the spinal cord, the conus and cauda equina and associated nerve roots typically results in paralysis, pain, sensory loss, and loss of bladder and bowel control. No successful treatments are presently available for these patients. Leif Havton has developed an injury model in which ventral root avulsion or tear lead to physiological and behavioral consequences that approximate those observed following trauma to the spinal cord in humans. He has also developed a repair strategy whereby lesioned nerve roots are surgically reimplanted into the cord away from the injury to reinnervate leg muscles and the bladder. A collaborative team including Geschwind and Steve Goldman are identifying new targets for therapeutic interventions in this disease, such as replacing motor neurons and delivering axon growth-promoting factors, in collaboration with the PNS group. Moreover, the APNRR has gathered a team of international neurosurgeons to devise a clinical study of the potential effectiveness of translating the ventral root repair strategy into patients at onset of paralysis.
Molecular Determinants of Neural Repair
Another APNRR collaborative strategy utilizes a multifaceted approach to determine how one can modulate a number of endogenous biological processes- simultaneous induction of a cassette of genes, modulation of cytosolic protein function that will act at cellular, local and systemic levels- to facilitate protection and repair in both the peripheral and central nervous system. A number of genetic and small molecule targets, such as HDAC inhibitors, arginase-I, inosine, Runx-3, phosphoserine, mGluR-4, NAD, and ATF-3 have been identified as possible regulators of neural repair and are being studied by Brett Langley, Rajiv Ratan, Benowitz, Groner, Harley Kornblum, He, and Clifford Woolf. These targets are utilized in a variety of in vitro repair model systems available to APNRR investigators (Tom Carmichael, Giger, Hoke, Benowitz, Havton) to evaluate their efficacy across models.
Proper nervous system function requires that ion channels be clustered and retained at specific locations in axons. For example, the initiation of electrical signals occurs at the axon initial segment and the propagation of action potentials in myelinated axons depends on clustered ion channels at nodes of Ranvier. Many nervous system diseases and injuries disrupt these functional axonal domains leading to conduction block. Since ion channel clustering is so critical for proper nervous system function, any therapeutic strategy aimed at neural repair and regeneration must include the maintenance and re-establishment of nodes and axon initial segments. In collaboration with Peles, Benowitz, Carmichael, Havton, and Goldman, Mathew Rasband is evaluating various APNRR models for the recovery of the functional organization of axons. With Barres, Peles, and Goldman, Rasband is also determining the mechanisms that control the molecular assembly and maintenance of nodes and axon initial segments using a monoclonal antibody that permits live labeling of axon initial segments of cultured neurons, axon clustering assays, and gene expression profiling.
Axonal Sprouting and Plasticity
Stroke induces at least two processes for neural repair that correlate with functional recovery. Neurons that survive the stroke in the peri-infarct cortex sprout new connections (Carmichael). Neural progenitor cells in the subventricular zone produce immature neurons that are directed to areas of damage, a process termed post-stroke neurogenesis. Axonal sprouting and directed neurogenesis after stroke are entirely new biological processes for the adult brain. Adult neurons must elaborate a growth program to support axonal sprouting and both neural progenitor cells and immature neurons must express genes that mediate a new and aberrant migration of immature neurons. The goals of this collaborative effort (Carmichael, Roman Giger, Fainzilber, Sofroniew, and Geschwind) are to identify the genes that control axonal sprouting and neurogenesis after stroke, identify the inhibitory molecules that limit these processes, and manipulate these and other adaptations over time to lessen sensory and motor impairments. Giger tests conditional neuropilin and nogo receptor knockout mice in order to define the functional interactions within growth inhibitory systems that regulate axonal sprouting and neuroblast migration/differentiation after stroke. Sofroniew and Tim Deming provide novel synthetic polypeptide biomaterials that serve as vehicles to deliver matrix molecules and growth factors to enhance post stroke regeneration. Blesch supplies bicistronic lentiviral constructs for the study of genes that are involved in post-stroke axonal sprouting, while Fainzilber will perform high throughput imaging on candidate axonal sprouting molecules that have been identified in Carmichael's single cell, post-stroke sprouting screen.
During normal adult neurogenesis neural progenitors go through a highly characteristic electrophysiological maturation that corresponds to their morphological maturation, in both the olfactory bulb and hippocampus. It is possible that a similar process may occur with neural progenitors as they develop in peri-infarct tissue after stroke. Istvan Mody performs brain slice recordings and morphological studies of identified neurons in Carmichael's focal stroke model, while Sofroniew generates transgenic mice to determine the electrophysiological properties of newly born neurons as they migrate in their immature state and after long-term survival and maturation in peri-infarct cortex. Furthermore, the process of recovery from injury may involve some of the same mechanisms activated during learning. Carmichael, James Conner, and Esther Shohami examine whether pathways can be activated after stroke and traumatic brain injury to accelerate recovery. To explore this hypothesis, Alcino Silva works with this group to determine whether a core group of molecular mechanisms altered in transgenic mutant smart mice with mutations that enhance learning abilities can lead to faster or more complete recovery after stroke and brain trauma.
Stem Cell Differentiation and Expression
Since their discovery in 1992, stem cells have held promise as therapeutic tools in a variety of neurologic diseases, such as stroke, spinal cord injury, amyotrophic lateral sclerosis (ALS), trauma and even brain tumors. Greater knowledge of the biology of stem cells and other obstacles must be overcome prior to utilizing them for meaningful repair. Carmichael found that stroke results in a mobilization of endogenous progenitors to become neurons, but the extent of this neurogenesis is relatively weak and short-lived. Methods to enhance the expression, differentiation into needed cell types, and survival of neurons from endogenous or exogenously administered neural stem cells need to be developed. Goldman, Havton, Kornblum, and Strickland are presently engaged in collaborations to address these and other issues required to make stem cells a viable therapeutic option. Goldman examines signal activation of glial progenitor cells after demyelinating injury and subsequent remyelination. In collaboration with Havton and Geschwind, Goldman is also looking into gene expression-predicted treatment strategies for motor neuronopathies in lumbosacral injury by utilizing stem cells under control of the Hb9 promoter, which is sufficient to direct gene expression specifically to motor neurons. In addition to screening small molecules to induce stem cell differentiation into neurons, Kornblum and Paul Mischel work with Goldman to identify differences in gene expression patterns between tumor progenitor cells and glial progenitor cells. This will help to identify signaling pathways that are specifically dysregulated during oncogenesis versus those that cause gliomagenesis. The Strickland lab has been successful in the application of mesenchymal adult stem cells to aid in recovery in PNS injury models. They will expand this further by partnering with the Heintz lab to label the stem cells and track their differentiation.
In addition to the highly collaborative nature of APNRR, core platforms are available to all of the investigators to foster the collaborative process. All data collected in each platform is freely available to any investigator within APNRR. An Imaging Platform run by John Mazziotta, Bruce Dobkin, Alger, and Arthur Toga includes access to MRI, PET, and functional MRI for small animal and human studies, as well as postmortem digital imaging and supercomputing. Optical intrinsic signal and fluorescence imaging (Andrew Charles) and 2-photon studies (Carlos Portera-Cailliau and others) allow investigators to track activity-dependent plasticity, changes in dendritic morphology, and synaptic plasticity. High throughput imaging allows rapid screening for neurite outgrowth on the level of millions of cells (Fainzilber). A novel cortical and subcortical stroke platform as well as relevant behavioral tests in mice (Carmichael lab) model human neurorehabilitative therapies, and evaluate candidate stroke repair molecules and cell-based therapies. Heintz has developed a novel platform that allows the purification of RNA from genetically labeled cells by the combination of BACarray technology and ribosome immunoprecipitation (RIParray). This information will provide the APNRR investigators fundamental insight into the gene expression that underlies and defines the role of the identified cell types. Blesch operates a lentiviral platform to produce recombinant lentivirus to express genes for project-specific applications within APNRR. A proteomics platform (Burlingame) enables investigators to identify proteins that work together to maintain normal cell function as well as to decipher the nature of molecules that underlie repair processes. Griffin oversees the axonal transport platform, which allows the detection of fluorescent particle movement to identify axon collateral regeneration. All microarray data is processed in an identical manner and stored in a centralized database through a platform (Geschwind and Coppola). Digital immune-electron microscopy, camera lucida, and other imaging of cellular organelles, synapses, remyelinating processes, and cell types is carried out by Havton's anatomical platform. Expertise in the design of translational clinical trials (Dobkin, Tuszynski, Havton, Hoke) allows an ongoing assessment of the relevance of findings from models and rapid progress toward phase 1 & 2 trials of interventions.
The Collaborative Process
These and new investigators who are invited to attend workshops continue to enrich each other's ideas and investigations and challenge the group to find robust treatments for disabled patients.